A large number of peptides have been approved for use in medical practice, and the peptides may be produced in suitable host cells by recombinant DNA technology or they may be produced synthetically by well established peptide synthesis technology. However, native peptides as well as analogues thereof tend to exhibit high clearance rates which are unacceptable for many clinical indications where a high plasma concentration of the peptide is required over a prolonged period of time.
A variety of derivatizations of peptides and peptide analogs have been found to influence the clearance rate of the peptides in a favourable direction as e.g. described in WO98/08871, WO98/08872, WO99/43708, EP 1227107 and WO00/55119. One such derivatization is the introduction of a lipophilic acyl group into the therapeutic peptide causing a desirable protracted profile of action relative to the non-acylated peptide. Hence, less frequent administration of the therapeutic protein improves the patients compliance to the prescribed therapy, and it reduces the amount of peptide to be administered.
In order for therapeutic peptides to be economically viable the cost of producing the peptides as well as the therapeutic dosage of the peptide are pivotal. A major cost during production of therapeutic peptides is the purification steps required to separate the target protein from impurities which are closely related to the target protein. These purification steps are usually performed by chromatography implying expensive chromatography matrices and solvents as well as reduced overall yield.
Introduction of an acyl group into one or more sites of a peptide or protein with more than one nucleophilic atom, such as more than one amine, can give rise to a product mixture of product-related impurities with great resemblance to the desired compound in the crude mixture which gives rise to loss of product and subsequently, a difficult purification step.
It is the aim of the present invention to provide an efficient, robust and economic method for the introduction of lipophilic groups into peptides or proteins via a spacer. Said spacer may e.g. be composed of one or more 8-amino-3,6-dioxaoctanoic acid moietie(s) and glutamic acid. The method is more specific, robust and thus results in higher yields and reduced formation of closely related impurities than other known methods. A significant reduction of the cost of producing the acylated peptides or proteins is achieved. Less expensive acylated peptides are highly desirable for maximizing the number of patients for whom the treatment is available as well as for exploiting the advantages of alternative delivery routes which have lower bioavailability than subcutaneous injection, e.g. transdermal, pulmonal or oral delivery.
The present invention thus provides a method for selectively acylating the epsilon-amino group of a lysine residue in a peptide or protein with surprisingly high selectivity.